The overall goal of this project is to investigate the pathogenesis of prion diseases using transgenic mouse models. Our objective is to understand the molecular and cellular mechanisms by which prions kill neurons and damage the central nervous system. During the last funding period, we investigated how three, distinct genetic mechanisms (loss, gain, and subversion of the normal function of PrPC) contribute to neurodegeneration in two different transgenic models: Tg(PG14) mice, which express an aggregation-prone PrP harboring a nine-octapeptide insertional mutation linked to familial Creutzfeldt-Jakob disease;and Tg(CR) mice, which express a highly neurotoxic form of PrP with a 21-amino acid deletion (residues 105-125) in the conserved, central region of the protein. In the course of our previous work, we made three key discoveries that form the basis for this renewal application. First, we found that mutations in the central region of PrP, including deletions such as CR, as well as point mutations associated with familial prion diseases of humans, induce a powerful ion channel activity that can be observed by patch-clamping techniques. Second, we have identified a group of PrP ligands that inhibit the ion channel activity of mutant PrP. Interestingly, most of these ligands also inhibit the formation of PrPSc, in cell-free systems or mice, suggesting that common structural domains control both the functional activity of PrP and its conversion to PrPSc. Third, we observed that a positively-charged, nine amino acid domain at the N-terminus plays an essential role in the channel-forming and neuroprotective properties of PrP molecules. Taken together, these new findings suggest the hypothesis that the pathogenesis of some inherited prion diseases, and possibly of infectiously acquired cases as well, may be due to PrP-related channel activity. Moreover, these pathogenic effects may be determined by a surprisingly short domain of the PrP primary sequence that controls both ion channel activity and conformational misfolding of the protein. To pursue these observations, we plan to (1) Analyze transgenic models of familial prion diseases associated with a "channel-inducing" PrP mutations;(2) Determine whether PrPSc induces PrPC-dependent, ion channel activation;(3) Test whether inhibitors of PrP-related ion channel activity have therapeutic effect in mouse models of familial and infectious prion diseases;and (4) Investigate the function of the N-terminal polybasic domain of PrP a critical neurotoxicity determinant. This project addresses a major gap in knowledge concerning the mechanisms by which prions are neurotoxic. It explores specific molecular and cellular pathways that may be activated by prions, and that may a role in their pathogenic effects. The proposal ties prion diseases to other neurodegenerative conditions due to abnormal activity of ion channels, and it sets the stage for treating prion diseases by blocking specific neurotoxic pathways in addition to prion propagation. PUBLIC HEALTH RELEVANCE: Prion diseases are fatal neurodegenerative disorders of humans and animals that pose a grave threat to public health, and endanger the safety of the food, blood and organ supplies. This grant application utilizes genetically engineered mice to explore the mechanisms by which prions kill nerve cells and damage the brain. The project sets the stage for development of novel therapeutic approaches based on blocking specific neurotoxic pathways.